Vector constructs

ABSTRACT

Improved vector constructs useful in the expression of double-stranded RNA are provided. The constructs are particularly useful for expression of double-stranded RNA in vitro and in vivo.

RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. 119 of Great Britain application number GB 0012233.3, filed May 19, 2000.

FIELD OF THE INVENTION

[0002] The invention relates to improved vector constructs for use in the expression of double-stranded RNA, particularly for use in the expression of double-stranded RNA in vitro and in vivo.

BACKGROUND OF THE INVENTION

[0003] Since the advent of double-stranded RNA inhibition (RNAi) as a tool for controlling gene expression, as described in WO 99/32619 and WO 00/01846, there has been recognized a need for specialized vectors designed for the production of double-stranded RNA (dsRNA).

[0004] Cloning vectors designed to produce high levels of dsRNA have been previously described by Plaetinck et al. (WO 00/01846) and Timmons et al. Nature, 395:854 (1998). These vectors generally contain a multiple cloning site (MCS) into which target DNA fragments can be cloned flanked by two opposable transcriptional promoters. Essentially, these three components (Promoter 1, MCS and Promoter 2) make up the entire system. In the appropriate expression system, the DNA cloned into the MCS may be transcribed in both directions, leading to the production of two complementary RNA strands.

[0005] A disadvantage of the known systems is that not only the cloned fragment is transcribed. Read-through of the RNA polymerase will result in transcription of the entire vector, and this also in both directions. As only transcription of the cloned DNA fragment will result in active dsRNA for RNAi purposes, transcription of the vector part results in useless, inefficient RNA. More specifically, 80% of these transcripts can be considered as non-specific and thus non-effective.

[0006] The large amounts of non-specific RNA generated by the prior art plasmid and expression systems results in some undesirable side effects. First, in RNAI protocols based on introduction of dsRNA into C. elegans via a food organism such as E. coli which expresses the dsRNA (see WO 00/01846), large RNA strands are considered to be toxic for the food organism. As a result, high amounts of RNA accumulating in E. coli cause a significant part of the population to die.

[0007] Second, and probably more important, is the reduction of inhibition potential. The presence of large amounts of non-specific dsRNA causes a competitive environment for the specified sequences. The potential of the template-specified dsRNA sequences to inhibit the targeted protein expression in, for instance, C. elegans cells is reduced by the presence of these large non-specific regions. Such an inhibition by non-specific dsRNA has also been shown in Drosophila by Tushl et al., Genes & Development 13:3191-3197 (1999). Not only the potential to inhibit gene expression is affected, but also the amount of specific dsRNA produced is limited.

[0008] Third, transcription of the vector backbone part, more particularly transcription of the origin of replication and related structures, results in plasmid instability and plasmid reorganisation, leading to reduced production of dsRNA. This relatively low concentration of effective dsRNA in turn leads to inefficient RNAi.

[0009] To conclude, the previously described vectors have following shortcomings: they are toxic to the feeding organism, a greater proportion of the transcripts produced are non-specific, the inhibitory potential of the dsRNA is reduced by the presence of non-specific regions, a high incidence of plasmid reorganizations and loss of plasmid from the feeding organism. It is therefore an object of the present invention to provide improved vectors for the production of dsRNA which avoid the disadvantages of the prior art vectors.

[0010] Vectors for use in the in vitro synthesis of RNA transcripts, for example the production of RNA probes, have been known and commonly used in the art for some time (see for example F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); Jendrisak et al, Vectors for in vitro production of RNA copies of either strand of a cloned DNA sequence, U.S. Pat. No. 4,766,072). In standard in vitro transcription protocols the problem of read-through transcription of vector sequences is generally avoided by linearizing the transcription vector at restriction site positioned at the 3′ end of the desired transcript. However, this solution is not appropriate for in vivo transcription or for the production of dsRNA where it is important that the template is transcribed in both directions.

SUMMARY OF THE INVENTION

[0011] The inventors now propose a novel solution to the problems encountered with the prior art vectors for the production of dsRNA, based on the use of transcription terminators. Generally the solution consists of the use of at least one transcription terminator operably linked to at least one promoter, wherein the terminator stops the transcription initiated by the promoter. Any DNA fragment inserted between the 3′ end of the promoter and the 5′ end of the terminator will then be transcribed, without the unwanted transcription of the vector backbone. Preferentially the vector consists of two promoters and two terminators, as further described below.

[0012] Therefore, in accordance with a first aspect of the invention there is provided a DNA construct comprising two opposable promoters flanking an inter-promoter region, the construct further comprising at least one transcription terminator positioned transcriptionally downstream of one of the said promoters. In particular, the invention provides for: a DNA construct comprising:

[0013] a) a first promoter and

[0014] b) a second promoter, in which the first and second promoter are in opposite orientation to each other and define:

[0015] c) an inter-promoter region positioned downstream of the 3′ end of the first promoter and downstream of the 3′ end of the second promoter; and which DNA construct further comprises:

[0016] d) at least one cloning site positioned in the inter-promoter region; and

[0017] e) a first transcription terminator, positioned (as seen from the 3′ end of the first promoter) downstream of the first promoter and downstream of the at least one cloning site, wherein the first transcription terminator is operably linked to the first promoter.

[0018] The inter-promoter region can also further be defined as: the DNA region between the 3′ end of the first promoter and the 3′ end of the second promoter, and which is downstream of the first promoter, and which is downstream of the second promoter, and which preferably does not contains the 5′ end of the first promoter and of the second promoter. The opposable first promoter and second promoter drive expression directional from their 5′ ends to their 3′ ends starting transcription downstream of their 3′ ends, thus providing transcription of both strands of any nucleotide sequence(s) present in the inter-promoter region.

[0019] The two promoters present in the DNA construct of the invention may be identical or they may be different and may be of essentially any type. The precise nature of the promoters used in the construct may be dependent on the nature of the expression system in which the construct is expected to function (e.g. prokaryotic vs eukaryotic host cell). Bacteriophage promoters, for example the T7, T3 and SP6 promoters, are preferred for use in the constructs of the invention, since they provide advantages of high level transcription which is dependent only on binding of the appropriate RNA polymerase. Each of these promoters can independently be chosen. The phage promoters can also function in a wide variety of host systems, i.e. both prokaryotic and eukaryotic hosts, provided that the cognate polymerase is present in the host cell.

[0020] The arrangement of two “opposable” promoters flanking an inter-promoter region such that transcription initiation driven by one of the promoters results in transcription of the sense strand of the inter-promoter region and transcription initiation driven by the other promoter results in transcription of the antisense strand of the inter-promoter region is an arrangement well known in the art, for example, in the pGEM7 series of vectors from Promega Corp., Madison Wis., USA.

[0021] The DNA constructs of the invention differ from those of the prior art because of the presence of at least one transcription terminator positioned transcriptionally downstream of one of the promoters. The transcription terminator may be uni- or bi-directional, the choice of uni- vs bi-directional terminators being influenced by the positioning of the terminator(s) within or outside the inter-promoter region, as explained below. The terminator may be of prokaryotic, eukaryotic or phage origin. Bacteriophage terminators, for example T7, T3 and SP6 terminators, are particularly preferred. The only requirement is that the terminator must be capable of causing termination of transcription initiating at the promoter relative to which it is transcriptionally downstream. In practice, these means that the promoter and terminator must form a ‘functional combination’, i.e. the terminator must be functional for the type of RNA polymerase initiating at the promoter. By way of example, a eukaryotic RNA pol II promoter and a eukaryotic RNA pol II terminator would generally form a functional combination. The selection of a functional combination is particularly important where bacteriophage promoters and terminators are to be used in the constructs of the invention, since the phage promoters and terminators are both polymerase-specific. To form a functional combination both the promoter and the terminator should be specific for the same polymerase, e.g. T7 promoter and T7 terminator, T3 promoter and T3 terminator etc.

[0022] In one embodiment, the DNA construct of the invention may comprise a single transcription terminator, positioned (as seen from the 3′ end of the first promoter) downstream of the first promoter and downstream of the at least one cloning site, wherein the first transcription terminator is operably linked to the first promoter, wherein the single transcription terminator is positioned in the inter-promoter region.

[0023] In an alternative arrangement, the DNA construct comprises a single transcription terminator positioned outside of the inter-promoter region. In a still further embodiment, the DNA construct may comprise two transcription terminators, each one of which is positioned transcriptionally downstream of one of the two promoters. In this arrangement, one or both of the terminators may be positioned within the inter-promoter region. These various embodiments of the DNA constructs of the invention will be more fully described below, with reference to the accompanying drawings. The position of a first transcription terminator outside the inter-promoter region may also be further defined as, i.e. such that a first transcription terminator is positioned (as seen from the 3′ end of the first promoter) downstream of the first promoter, downstream of the at least one cloning site, and downstream of the 5′ end of the second promoter.

[0024] The position of a second transcription terminator outside the inter-promoter region may also be further defined as, i.e. such that a second transcription terminator positioned (as seen from the 3′ end of the second promoter) downstream of the second promoter, downstream of the at least one cloning site, and downstream of the 5′ end of the first promoter.

[0025] Moreover, when the terminator is not located in the inter-promoter region, the distance between the 5′ end of the first promoter and 3′ end of the second terminator, or the distance between the 5′ end of the second promoter and the 3′ end of the first terminator is preferably small, i.e. such that the 3′ end of the first transcription terminator is separated from the 5′ end of the second promoter by no more than 2000 nucleotides, preferably no more than 1000 nucleotides, more preferably no more than 500 nucleotides, even more preferably no more than 200 nucleotides, especially preferably no more than 100 nucleotides, more especially preferable no more than 50 nucleotides, even more especially preferably no more than 20 nucleotides, particularly preferably no more than 10 nucleotides, more particularly preferably no more than 6 nucleotides.

[0026] Furthermore, when the second transcription terminator is located outside of the inter-promoter region, preferably the 3′ end of the second transcription terminator is separated from the 5′ end of the first promoter by no more than 2000 nucleotides, preferably no more than 1000 nucleotides, more preferably no more than 500 nucleotides, even more preferably no more than 200 nucleotides, especially preferably no more than 100 nucleotides, more especially preferably no more than 50 nucleotides, even more especially preferably no more than 20 nucleotides, particularly preferably no more than 10 nucleotides, more particularly preferably no more than 6 nucleotides.

[0027] As defined above the term ‘inter-promoter region’ refers to all of the DNA sequence between the two promoters. As explained above, in certain embodiments of the invention the transcription terminator(s) may be sited within the inter-promoter region. The inter-promoter region may, advantageously, comprise a sequence of nucleotides forming a template for dsRNA production. The precise length and nature of this sequence is not material to the invention. The invention further provides DNA constructs in which the inter-promoter region comprises a cloning site. The function of the cloning site is to facilitate insertion of a DNA fragment forming a template for dsRNA production between the two promoters. Thus, the invention provides a series of cloning vectors which are of general use in the construction of template vectors for dsRNA production. Also encompassed within the scope of the invention are vectors derived from the cloning vectors which have a DNA fragment inserted into the cloning site.

[0028] The cloning site may further comprise one or more of the following:

[0029] at least one restriction site,(as known in the art), or one or more further restriction sites, e.g. to provide a multiple cloning site(as known in the art),

[0030] a stuffer DNA, e.g., flanked by at least two restriction site, such as two BstXI restriction sites, or two XcmI restriction sites,

[0031] attR1 and attR2 recombination sites,

[0032] a ccdB nucleotide sequence,

[0033] a ccdB nucleotide further comprising at least one unique blunt-end restriction site, such as a SrfI restriction site, and/or

[0034] a DNA fragment inserted in the at least one cloning site.

[0035] All of the DNA constructs provided by the invention may, advantageously, form part of a replicable cloning vector, such as, for example, a plasmid vector. In addition to the opposable promoters, inter-promoter region and transcription terminator(s), the vector ‘backbone’ may further contain one or more of the general features commonly found in replicable vectors, for example an origin of replication to allow autonomous replication within a host cell and a selective marker, such as an antibiotic resistance gene. The selective marker gene (e.g. the antibiotic resistance gene) may itself contain a promoter and a transcription terminator and it is to be understood that these are completely independent of the promoter and terminator elements required by the invention and are not to be taken into consideration in determining whether a particular vector falls within the scope of the invention.

[0036] DNA constructs according to the invention may be easily be constructed from the component sequence elements using standard recombinant techniques well known in the art and described, for example, in F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994), as will be appreciated by one skilled in the art from the following detailed description of the invention and the accompanying Examples.

[0037] There follows a detailed description of DNA constructs according to the invention, with reference to the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1(a) to 1(e) are schematic representations of several different embodiments of the DNA construct according to the invention illustrating the relative positioning of the promoter and transcription terminator elements.

[0039]FIG. 2(a) is a schematic representation of a prior art vector included for comparison purposes.

[0040] FIGS. 2(b) to 2(e) are schematic representations of several further embodiments of the DNA construct according to the invention illustrating the use of different cloning sites in the inter-promoter region.

[0041]FIG. 3 is a representation (plasmid map) of pGN1.

[0042]FIG. 4 is a representation (plasmid map) of pGN9.

[0043]FIG. 5 illustrates the nucleotide sequence of a fragment of plasmid pGN1 (SEQ ID NO:1), annotated to show the positions of the opposable T7 promoters.

[0044]FIG. 6 depicts the nucleotide sequence of the T7 transcription terminator (SEQ ID NO:2).

[0045]FIG. 7 illustrates the sequences of oligonucleotides oGN27 (SEQ ID NO:3), oGN28 (SEQ ID NO:4), oGN29 (SEQ ID NO:5) and oGN30 (SEQ ID NO:6) used to insert T7 transcription terminators into pGN1. The positions of the T7 pol terminator sequences and of various restriction sites are marked.

[0046]FIG. 8 illustrates the nucleotide sequence of a fragment of plasmid pGN9 (SEQ ID NO:7), annotated to show the positions of the opposable T7 promoters (T7p) and the T7 transcription terminators (T7 term).

[0047]FIG. 9(a) is a representation (plasmid map) of pGN29; FIG. 9(b) is a representation (plasmid map) of pGN39; FIG. 9(c) is a representation (plasmid map) of the plasmid TopoRNAi.

[0048]FIG. 10 shows the complete nucleotide sequence of plasmid pGN9 (SEQ ID NO:8).

[0049]FIG. 11 shows the complete nucleotide sequence of plasmid pGN29 (SEQ ID NO:9).

[0050]FIG. 12 shows the complete nucleotide sequence of plasmid pGN39 (SEQ ID NO:10).

[0051]FIG. 13 shows the complete nucleotide sequence of plasmid TopoRNAi (SEQ ID NO:11).

[0052]FIG. 14 shows the complete sequence of plasmid pGN49A (SEQ ID NO:12).

[0053]FIG. 15 shows the complete sequence of plasmid pGN59A (SEQ ID NO:13).

[0054]FIG. 16 is a representation (plasmid map) of pGN49A.

[0055]FIG. 17 is a representation (plasmid map) of pGN59A.

DETAILED DESCRIPTION OF THE INVENTION

[0056] Referring to the Drawings, FIG. 1(a) schematically illustrates a first DNA construct according to the invention which is a plasmid vector comprising two opposable promoters; a first promoter (a) and a second promoter (b) flanking an inter-promoter region (c), which inter-promoter region is downstream of the 3′ of the first promoter, and down stream of the 3′ end of the second promoter. The first promoter and the second promoter may be identical or different. This embodiment comprises a first transcription terminator (e) and a second transcription terminator (f), both of which are positioned within the inter-promoter region. In this embodiment, the first terminator and the second terminator are preferentially uni-directional terminators.

[0057] A DNA fragment may be inserted in the at least one cloning site (d). Such DNA fragment is subject to transcription directed by the first promoter (a) and the second promoter (b) (i.e. transcription of both strands), resulting in the generation of two RNA fragments which may combine to form double-stranded RNA of the inserted DNA fragment (both in vitro and in vivo).

[0058] Any desired DNA sequence, such as a genomic DNA sequence, or a cDNA sequence or any other coding sequence, may be inserted in the at least one cloning site. Without being limited to any specific explanation, it is assumed that when (a) and (e) form a functional combination, RNA polymerase which initiates transcription at (a) will transcribe the inter-promoter region including the at least cloning site and the DNA fragment inserted in the at least cloning site and will be terminated when it reaches (e). Similarly, RNA polymerase which initiates transcription at (b) will transcribe the inter-promoter region including the at least one cloning site and the DNA fragment inserted in the at least one cloning site and will terminate when it reaches (f). The terminators cause the RNA polymerase to pause, stop transcription and fall off the template. This prevents the unlimited transcription of the vector backbone, and reduces the unspecific transcription of non-essential DNA.

[0059] The inter-promoter region further comprises a sequence of nucleotides corresponding to a target for double-stranded RNA inhibition. This sequence is designated ‘TF’=for target fragment. It is this sequence which, when transcribed into dsRNA, will be responsible for specific double-stranded RNA inhibition of a target gene. The target fragment may be formed from a fragment of genomic DNA or cDNA from the target gene. Its precise length and nucleotide sequence are not material to the invention.

[0060] In the arrangement shown in FIG. 1(a) the two terminators are positioned on either side of the TF within the inter-promoter region. Each of the terminators is positioned transcriptionally downstream of one of the promoters, the fist terminator (e) is transcriptionally downstream of first promoter (a) and the second terminator (f) is transcriptionally downstream of the second promoter (b). Assuming that (a) and (e) form a functional combination, as described above, RNA polymerase which initiates transcription at a) will transcribe the inter-promoter region up to and including TF and will be terminated when it reaches (e). Similarly, RNA polymerase which initiates transcription at (b) will transcribe the inter-promoter region up to and including TF on the opposite strand and will terminate when it reaches (f). The terminators cause the RNA polymerase to pause, stop transcription and fall off the template. This prevents the unlimited transcription of the vector backbone, and reduces the unspecific transcription of non-essential DNA.

[0061] The transcripts generated from this vector may, depending on the precise placement of the terminators in the vector, be almost completely specific dsRNAs corresponding to the TF region. Through the direct placement of the terminator sequences at the downstream end of the TF region on both sides of the inserted DNA fragment, the amount of material transcribed is completely reduced to the template-specified sequences. Therefore, a higher amount of specific dsRNA is obtained. Furthermore the constructs are now also more stable, due to the non-transcription of the vector backbone. The latter characteristic (stability), combined with the now relatively higher specific transcription rate, provides a system adapted to synthesize higher amounts of specific short dsRNA strands. This proportionally higher amount of transcript, resulting in high concentrations of specific dsRNA, enhances the inhibitory effect in RNAi protocols. In RNAi protocols based on expression of dsRNA in a food organism, toxicity for the feeding organisms due to high RNA expression is brought to a minimal level by use of this vector.

[0062] A specific example of a vector of the type illustrated in FIG. 1(a), considered by the inventors to be the optimal arrangement for RNAi applications, is plasmid pGN9 described in the accompanying Examples. The transcriptional terminators used in pGN9 are T7 RNA polymerase specific terminators, since the vector contains two opposable T7 promoters. However, other systems could be used such as an expression system based on the T3 or SP6 promoter, terminator and polymerase or other prokaryotic or eukaryotic promoters and terminators.

[0063]FIG. 1(b) illustrates schematically a further DNA construct according to the invention which is a plasmid vector comprising two opposable promoters (a) and (b) flanking an inter-promoter region (c). This vector also comprises two transcription terminators (e) and (f) but in this arrangement the two terminators are positioned outside of the inter-promoter region, in fact the terminator elements now flank the two promoters. The arrangement is such that (e) is transcriptionally downstream of (a) whilst (f) is transcriptionally downstream of (b). Here again (e) terminates the transcription initiated by (a), whilst (f) terminates the transcription initiated by promoter (b). Placement of the terminators outside of (d) allows the use of bi-directional terminators as well as uni-directional terminators, in contrast to the arrangement in FIG. 1(a) where uni-directional terminators are preferred because of the placement of the terminators between the promoters. A number of bi-directional terminators which could be used in accordance with the invention are known in the art. Generally these are observed to be polymerase non-specific.

[0064] The embodiment shown in FIG. 1(b) provides essentially the same advantages as that shown in FIG. 1(a) over the prior art vectors for dsRNA production. The vector shown in FIG. 1(b) will lead to the production of transcripts which are slightly longer, including the promoter regions. This relatively small difference in the length of the transcript and hence the formed dsRNA will not severely affect the efficacy in an RNAi system.

[0065] The position of the terminators and promoter in the example as shown in FIG. 1(b) are preferably placed at close proximity, such that the 5′ end of the promoters are separated from the 3′ end of the transcription terminators by no more than 2000 nucleotides, preferably no more than 1000 nucleotides, more preferably no more than 500 nucleotides, even more preferably no more than 200 nucleotides, especially preferably no more than 100 nucleotides, more especially preferably no more than 50 nucleotides, even more especially preferably no more than 20 nucleotides, particularly preferably no more than 10 nucleotides, more particularly preferably no more than 6 nucleotides.

[0066]FIG. 1(c) illustrates schematically a further DNA construct according to the invention which is a plasmid vector comprising two opposable promoters (a) and (b) flanking an inter-promoter region (c). In this embodiment one terminator (in this case (f)) is positioned within the (c) and the other (e) is positioned outside (c). Again, (e) terminates transcription initiated by (a) and (f) terminates transcription initiated by (b). This arrangement may provide a useful solution to the problem of one of the terminators interfering with polymerase activity in the other direction (e.g. (f) interferes with (b) initiated transcription). This vector essentially provides the same advantages as the vector variations shown in FIG. 1(a) and FIG. 1(b) over the prior art. The relatively small difference in the length of the transcript due to the transcription of one of the promoters will not significantly affect the efficacy in RNAi systems. This more particularly the case when the terminator that is located outside of the inter-promoter region (c) is at close proximity of the promoter, as defined above.

[0067] FIGS. 1(d) and 1(e) illustrate schematically two further DNA constructs according to the invention which are both plasmid vectors comprising two opposable promoters (a) and (b) flanking an inter-promoter region (c). These embodiments comprise a single terminator only. In the arrangement shown in FIG. 1(d) a single terminator (e) which terminates transcription from (a) is placed outside of (c). The placement of the terminator outside of the inter-promoter region allows the use of both a bi-directional terminator or a uni-directional terminator in this system. In the embodiment shown in FIG. 1(d), (e) is placed within the (c) and should therefore preferably be a uni-directional terminator.

[0068] Further embodiments of the DNA construct according to the invention are illustrated schematically in FIGS. 2(b) to 2(e).

[0069] These embodiments are all plasmid cloning vectors, based upon the optimal arrangement of promoters and terminators shown in FIG. 1(a), and described above, containing cloning sites to facilitate the insertion of a DNA fragment into the at least one cloning site.

[0070] These embodiments are all plasmid cloning vectors, based upon the optimal arrangement of promoters and terminators shown in FIG. 1(a), containing cloning sites to facilitate the insertion of a target DNA fragment into the inter-promoter region.

[0071]FIG. 2(a), which is a schematic representation of a prior art cloning vector, is included for comparison purposes. This vector comprises two opposable promoters (a) and (b), which may be identical or different, flanking a multi-cloning site (MCS).

[0072]FIG. 2(b) illustrates a first type of plasmid cloning vector according to the invention. The vector contains a first opposable promoter (a) and a second opposable promoter (b) flanking an inter-promoter region. The inter-promoter region can further be defined as: the DNA region between the 3′ end of the first promoter and the 3′ end of the second promoter, and which is downstream of the first promoter, and which is downstream of the second promoter, and which preferably does not contain the 5′ end of the first promoter and of the second promoter. The inter-promoter region further comprises terminators (e) and (f) flanking a multi-cloning site MCS. The MCS comprises at least one individual restriction site, and preferably more than one restriction site as known in the art, any of which may be used for insertion of a DNA fragment.

[0073]FIG. 2(c) illustrates a further type of plasmid cloning vector according to the invention. This vector again contains opposable promoters (a) and (b) flanking an inter-promoter region comprising terminators (e) and (f). In this embodiment, (a) and (b) flank a cloning site which is adapted for facilitated cloning of PCR fragments, comprising a stuffer DNA flanked by two identical restriction sites, in this case BstXI sites. The specific sequence of the stuffer DNA is not essential, provided that said stuffer DNA does not interfere with the desired effect and/or the desired activity of the DNA constructs of the invention. A specific example of a vector according to this aspect of the invention described herein is plasmid pGN29.

[0074] The cloning of PCR products using BstXI recognition sites and BstXI adaptors is generally known in the art. The BstXI adaptors are commercially obtained, such as from Invitrogen (Groningen, the Netherlands). These adaptors are non-palindromic adapters designed for easier and efficient cloning of PCR products into vectors. These use of these adaptors reduces the concatamerization of adapters or insert DNA by having non-complementary (CACA) overhangs. The stuffer DNA is included merely to allow easy differentiation between BstXI cut and uncut vector on the basis of size. Its precise length and sequence are not of importance.

[0075]FIG. 2(d) illustrates a further type of plasmid cloning vector according to the invention. This vector again contains opposable promoters (a) and (b) flanking an inter-promoter region comprising terminators (e) and (f). In this embodiment, (a) and (b) flank a cloning site which facilitates “High Throughput” cloning based on homologous recombination rather than restriction enzyme digestion and ligation. As shown schematically in FIG. 2(d), the cloning site comprises attR1 and attR2 recombination sites from bacteriophage lambda flanking a gene which is lethal to E. coli, in this case the ccdB gene.

[0076] An alternative cloning method of DNA fragments into this vector,(not shown in FIG. 2(d)), consists of a variant of this vector, wherein the ccdB DNA sequence further comprises at least one unique restriction site, preferably the at least unique restriction site is a blunt-end restriction site, such as a SrfI restriction site. Insertion of a DNA fragment in the at least one unique restriction site, results in inactivation of the ccdB gene, and hence in inactivation of the lethal ccdB gene product.

[0077] A further variant of a vector a shown in FIG. 2(d) in which the attr1 and the attR2 are not present. Such a vector comprises at least one cloning site, said at least one cloning site consisting of a ccdB sequence, said ccdB sequence further comprising at least one unique restriction site, preferably the at least unique restriction site is a blunt-end restriction site, such as a SrfI restriction site. Insertion of a DNA fragment in the at least one unique restriction site, results in inactivation of the ccdB gene, and hence in inactivation of the lethal ccdB gene product.

[0078] These cloning sites comprising the ccdB nucleotide sequence and/or the attR sites (R1 and/or R2) are derived from the Gateway™ cloning system commercially available from Life Technologies, Inc. The Gateway™ cloning system has been extensively described by Hartley et al. in WO 96/40724 (PCT/US96/10082). A specific example of a vector according to this aspect of the invention described herein is pGN39.

[0079]FIG. 2(e) and 2(f) illustrate a still further type of plasmid cloning vector according to the invention. This vector again contains opposable promoters (a) and (b) flanking an inter-promoter region (c) comprising terminators (e) and (f). In the embodiment shown in FIG. 2(e), terminators (e) and (f) flank a cloning site which facilitates “high throughput” cloning of PCR products by TA™ cloning. This cloning site comprises a stuffer DNA flanked by two identical restriction sites for an enzyme which generates overhanging T nucleotides. In this case the restriction sites are XcmI sites, but other sites which are cleaved to generate overhanging T nucleotides could be used with equivalent effect. The overhanging T nucleotides facilitate cloning of PCR products which have a overhanging A nucleotide. This principle is known as TA™ cloning. The cut vector with overhanging T nucleotides can be “topomerized” to generate a cloning vector of the type shown schematically in FIG. 2(f), by linking the enzyme topoisomerase to the overhanging T nucleotides. The resulting vector also facilitates the cloning of PCR products by the principle known as TOPO™ cloning.

[0080] Both the TOPO™ and TA™ cloning systems, although not for the vectors described in this invention, are commercially available from Invitrogen. The TOPO™ cloning system has extensively been described by Shuman in WO 96/19497 (PCT/US95/16099). The TA™ cloning system has extensively been described by Hernstadt et al. in WO 92/06189 (PCT/US91/07147).

[0081] It will be readily appreciated by the skilled reader that whilst FIGS. 2(b)-2(f) illustrate the inclusion of different cloning sites into a vector of the type illustrated in FIG. 1(a), these cloning sites could be included into any of the DNA constructs of the invention, including those illustrated schematically in FIGS. 1(b) to 1(e)

Application of the DNA Constructs of the Invention in RNAi Technology.

[0082] As aforementioned, a major application of the DNA constructs/vectors of the invention is in the production of double stranded RNA for use in RNAi technology. In particular, the constructs are useful in in vivo RNAi protocols in the nematode worm C. elegans.

[0083] In C. elegans, RNAi has traditionally been performed by injection dsRNA into the worm. Fire et al. describes these methods extensively in International Application No. WO 99/32619. In short, both strands of RNA are produced in vitro using commercially available in vitro transcription kits. Both strands of RNA are allowed to form dsRNA, after which the dsRNA is injected into C. elegans.

[0084] The new vector system developed by the present inventors is a drastic improvement on this traditional method. First, the RNA can be produced in one step, for instance by using two identical promoters such as in the vector pGN9. Second, and more important, due to the presence of terminators the transcripts, and hence the formed dsRNA, will be more specific as only the cloned target fragment will be transcribed. This will result in a more efficient RNAi.

[0085] A further method to perform RNAi experiments in C. elegans has been described by Plaetinck et al. in WO 00/01846. In this method C. elegans worms are fed with bacteria which produce dsRNA. The dsRNA passes the gut barrier of the worm and induces the same RNAi as if the dsRNA has been injected. For these experiments, the preferred E. coli strain is HT115 (DE3), and the preferred C. elegans strain is nuc-1;gun-1. The improved vectors provided by the invention also improve the efficiency of RNAi in this method, as shown in the example below, as only effective dsRNA is produced.

[0086] Another method to perform RNAi has also been described by Plaetinck et al. in WO 00/01846. In short, this method is based on the production of dsRNA in the worm itself. This can be done by using worm promoters in the described vectors, or by using a transgenic worm expressing a polymerase specific for non-C. elegans promoters present in the vector, such that this polymerase drives transcription of the dsRNA. The promoters will preferentially be selected from the known bacteriophage RNA promoters, such as T7 or T3 or SP6 RNA promoters, which provide the advantage of a high level of transcription dependent only on the binding of the cognate polymerase.

[0087] Plasmid vector DNA can be introduced into the worm by several methods. First, the DNA can be introduced by the traditional injection method (Methods in Cell Biology, Vol 48, C. elegans Modern Biological Analysis of an organism, ed. by Epstein and Shakes). Second, the DNA can be introduced by DNA feeding. As has been shown by Plaetinck et al. in WO 00/01846, plasmid DNA can be introduced into the worm by feeding the worm with an E. coli strain that harbors the plasmid. Preferentially the E. coli strain is OP50, or MC1061 or HT115 (DE3) but any other strain would suit for this purpose. The C. elegans strain is preferentially a nuc-1 mutant strain or a nuc-1; gun-1 strain. The plasmid DNA from the E. coli passes the gut barrier and is introduced into the nematode, resulting in the expression of dsRNA. As with the other RNAi methods described above, the use of the new vector system will enhance the RNAi by the production of only specific dsRNA.

[0088] The invention will be further understood with reference to the following experimental Examples, together with FIGS. 3-17.

EXAMPLES Example 1 Vector Construction

[0089] The starting point for construction of the vectors exemplified herein was plasmid pGN1. This plasmid, described in the applicant's co-pending International Application No. WO 00/01846, contains two opposable T7 promoters flanking a multi-cloning site.

[0090] Vector construction was carried out according to standard molecular biology techniques known in the art and described, for example, in F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0091] 1) Construction of pGN9

[0092] pGN1 was first digested with restriction enzymes EcoRI and KpnI. Oligonucleotides oGN27 (SEQ ID NO:3) and oGN28 (SE ID NO:4) (FIG. 7) were annealed to generate a double stranded fragment which was then ligated into the EcoRI/KpnI cut vector. The resulting plasmid was re-digested with XbaI and HindIII. Oligonucleotides oGN29 (SEQ ID NO:5) and oGN30 (SEQ ID NO:6) were annealed to generate a double-stranded fragment which was then annealed into the XbaI/HindIII cut vector. The resulting vector was designated pGN9 (FIGS. 4 and 10; SEQ ID NO:8).

[0093] 2) Construction of Further Cloning Vectors

[0094] pGN29 (FIG. 9(a); FIG. 11; SEQ ID NO:9) was generated by replacing the MCS in pGN9 with a stuffer DNA flanked by BstXI sites. BstXI adapters are commercially available from Invitrogen (Groningen, the Netherlands).

[0095] pGN39 (FIG. 9(b); FIG. 12; SEQ ID NO:39) was generated by following steps; pGN29 was digested with BsTXI. BstXI adapters (Invitrogen, Groningen, The Netherlands) were ligated to Cassette A provided by the GATEWAY™ system (Life Technologies, Inc.). Cassette A contains attR1, CmR, ccda, ccdB, attR2. The Cassette A with the adapters were then ligated into the digested pGN29, resulting in pGN39A. pGN39A contains a unique SrfI site in the ccdB gene.

[0096] The TopoRNAi vector (FIG. 9(c); FIG. 13; SEQ ID NO:11) was generated in the following way; pGN29 was digested with BstXI. Using PCR with the primers oGN103 (SEQ ID NO:14) and oGN104 (SEQ ID NO:15) and template pCDM8 (Invitrogen, Groningen, The Netherlands), a stuffer was generated (SEQ ID NO: 20) which includes XcmI sites. Onto the PCR product, BstXI adapters were ligated, and the resulting ligation product was ligated in the BstXI digested pGN29 vector resulting in the TopoRNAi vector. oGN103 (SEQ ID NO:14): 5′ TACCAAGGCTAGCATGGTTTATCACTGATAAGTTGG 3′ oGN104 (SEQ ID NO:15): 5′ TACCAAGGCTAGCATGGGCCTGCCTGAAGGCTGC 3′

[0097] pGN49A (SEQ ID NO:12) was constructed to insert an additional unique non-blunt restriction site and to delete the CmR gene from pGN39. Overlap PCR was used. A first PCR was performed with primers oGN126 (SEQ ID NO:16)and oGN127 (SEQ ID NO:17) and PGN39A as template. Using primers oGN128 (SEQ ID NO:18) and OGN129 (SEQ ID NO:19) and the same template a second fragment was generated. Overlap PCR using the resulting fragments and primers oGN126P and oGN129P resulted in a final PCR product. To this final PCR product, BstXI adapters were ligated, and the ligation product was ligated into pGN29 digested with BstXI. The resulting vector was designated pGN49A.

[0098] A control vector was generated to test the efficiency of the pGN49A cloning vector, such vector should contain the T7 promoters, but not the T7 terminators. For this, the XbaI insert of pGN49A was isolated and cloned in pGN1 digested with the same restriction enzyme. The resulting vector was designated pGN59A (SEQ ID NO:13). oGN126 (SEQ ID NO:16): pGATCTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGC oGN127 (SEQ ID NO:17): GGAGACTTTATCGCTTAAGAGACGTGCACTGGCCAGGGGGATCACC oGN128 (SEQ ID NO:18): CCAGTGCACGTCTCTTAAGCGATAAAGTCTCCCGTGAACTTTACCCGGTGG oGN129 (SEQ ID NO:19) pGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAG

EXAMPLE 2 To Illustrate the Usefulness of the Improved Vectors in RNA.

[0099] This experiment was designed to illustrate the improved efficiency of the improved vectors of this invention in double-stranded RNA inhibition, as compared to the vectors known from the prior art. A significant increase on the efficacy of the system could be expected, as more effective dsRNA was produced and hence RNAi performed better. The experimental system for this proof of concept experiment was to measure C. elegans rescue at 25° C. in nuc-1/pha-1(e2123)ts C. elegans mutants by RNAi of sup35 using dsRNA feeding of pGN-2 (−terminator) and pGN-12 (+terminator), with PGN-1 (empty vector) as a control and dilutor. The pha-1 ts/sup-35 mutation has extensively been described by Schnabel in WO 99/49066.

[0100] The nuc-1 mutation in C. elegans provides for a C. elegans strain exhibiting better uptake abilities, such as the uptake of the dsRNA complexes than wild type C. elegans. This mutant is deleted in the major DNAse enzymes, and inventors have proven in earlier co-pending applications that this C. elegans strain results in enhanced RNAi by feeding the nematode with dsRNAs.

[0101] The pha-1(e2123)ts mutation provides a mutant C. elegans strain with a phenotype of survival at 15° C. and lethality at 25° C. This lethality is suppressible by the inhibition of sup-35 expression. RNAi of sup-35 should thus facilitate the rescue of pha-1(e2123)ts at 25° C. The vectors of the present invention, when expressing dsRNA from sup-35, should increase the efficacy of sup-35 RNAi in rescuing pha-1(e2123)ts mutants at 25° C., compared to vectors that do not contain the terminators.

[0102] Vector pGN1 was used as empty vector. Vector pGN2 (−terminator) is a vector harboring sup-35 DNA and expressing sup-35 dsRNA when introduced in the appropriate host, the vector has no transcriptional terminators inserted. Vector pGN12 (+terminator) is a vector as described above, containing the transcriptional terminators, and hence resulting in improved dsRNA production when introduced into an appropriate host. Thus, this vector has two unidirectional transcriptional terminators, both placed inside of the inter-promoter region, and flanking the sup-35 fragment. Use of the latter vector was expected to increase the efficacy of the system, here meaning a better rescue (survival) of pha-1(e2123)ts mutants at 25° C.

[0103] Experimental Conditions

[0104] 12-well micro-titer plates were filled with approximately 2 ml of NGM agar per well. (1 liter of NGM agar: 15 g Agar, 1 g pep tone, 3 g NaCl, 1ml cholesterol solution (5 mg/ml in EtOH), with sterile addition after autoclaving of 9.5 ml 0.1M CaCl₂, 9.5 ml 0.1 ml MgSO₄, 25 ml 1M KH₂PO₄/K₂HPO₄ buffer pH 6, ampicillin (100 μg/l), 5 ml 0,1 M IPTG and 5 ml nystatin solution (dissolved 10 mg/ml in 1:1 EtOH:CH₃COONH₄7.5 M)

[0105] The dried plates were spotted with approximately 50 μl of an overnight culture of bacteria HT115 (DE3) (Fire A, Carnegie Institution, Baltimore, Md.) transformed with the plasmids. Individual nematodes at the L4 growth stage were then placed in single wells at day 1. In each well 1 nematode was placed (P1). At day two, the P1 nematodes were placed to a new well and left to incubate for a day. The same procedure was repeated at day 3. All plates were further incubated at 25° C. to allow offspring to be formed. Sup35 RNAi induced survival (rescue) as was measured by counting the offspring.

[0106] Results

[0107] RNAi experiment in C. elegans nuc-1/pha-1(e2123)ts mutants by feeding with E. coli expressing sup-35 dsRNA.

[0108] Set Up:

[0109] pGN1 as control

[0110] pGN2 (sup 35−Term.)

[0111] pGN12 (sup 35+Term.)

[0112] pGN2+pGN1 dilutions ½, ¼, ⅛, {fraction (1/16)}, {fraction (1/32)}

[0113] pGN12+pGN1 dilutions ½, ¼, ⅛, {fraction (1/16)}, {fraction (1/32)}

[0114] Conditions:

[0115] Incubation temperature 25° C.

[0116] Readout: Count offspring (adult hermaphrodites) pGN1 (control) Day 1 0 0 0 0 Day 2 0 0 0 0 Day 3 0 0 0 0 pGN2 (undiluted) pGN12 (undiluted) Day 1 12 4 48 32 Day 1 16 29 37 14 Day 2 24 23 80 85 Day 2 27 22 57 2 Day 3 5 0 9 16 Day 3 1 2 4 1 pGN 2 + 1, ½ dilution pGN 12 + 1, ½ dilution Day 1 0 7 0 2 Day 1 22 28 103 61 Day 2 9 10 0 3 Day 2 36 45 53 40 Day 3 0 2 0 0 Day 3 3 3 25 1 pGN 2 + 1, ¼ dilution pGN 12 + 1, ¼ dilution Day 1 28 23 0 0 Day 1 * 6 36 5 Day 2 6 3 0 0 Day 2 24 55 3 Day 3 0 0 0 0 Day 3 pGN 2 + 1, ⅛ dilution pGN 12 + 1, ⅛ dilution Day 1 0 0 4 0 Day 1 31 12 16 38 Day 2 0 0 11 0 Day 2 4 5 37 4 Day 3 0 0 0 0 Day 3 0 0 2 1 pGN 2 + 1, {fraction (1/16)} dilution pGN 12 + 1, {fraction (1/16)} dilution Day 1 0 0 0 0 Day 1 1 0 0 0 Day 2 0 0 0 1 Day 2 2 0 0 1 little Day 3 0 0 0 0 Day 3 0 1 1 1 pGN 2 + 1, {fraction (1/32)} dilution pGN 12 + 1, {fraction (1/32)} dilution Day 1 0 0 0 0 Day 1 0 0 1 0 Day 2 0 0 0 0 Day 2 0 L2 3 0 Day 3 0 0 0 0 Day 3 2 0 L3-L4 0

[0117] Conclusions

[0118] As expected, worms fed by bacteria harboring pGN1, did not result in the viable offspring, due to the lethal effect of the pha-1 mutation at this temperature. Feeding the nematodes with E. coli harboring pGN2 or pGN12 both result in viable offspring. This is due to the feeding of the worm with dsRNA from sup-35. The remarkable difference between the two feeding experiments can be seen in the dilution series. When diluting the bacteria harboring pGN2 with bacteria harboring pGN1, the number of offspring diminishes drastically, even at a low dilution of one to two. This dilution series indicates that high levels of dsRNA are needed to have a proper RNAi induction. In the feeding experiment with bacteria harboring pGN12, significant offspring is still observed at a dilution of one to eight. This indicates that in the bacteria harboring pGN12, much more effective dsRNA is formed.

[0119] This experiment clearly shows that the addition of terminator sequences in vectors to express dsRNA as described above provide a significant advantage in the generation of RNAi.

EXAMPLE 3 Comparison of RNAi Efficiency of Vectors with and without T7 Terminators(pGN49 vs pGN59)

[0120] Three different genes have been cloned in the vectors pGN49A and pGN59A. The cloning was performed by amplifying the gene fragments with PfuI DNA polymerase producing blunt ends, facilitating cloning in these vectors. These PCR fragments were cloned into the vectors digested with SrfI. Correct fragment insertion of the clones was checked by PCR. The fragments are chosen such that ds expression and RNAi results in a lethal phenotype of the offspring. This procedure allows fast and easy comparison of the efficiency of the two vectors pGN49 and pGN59 in RNAi. plasmid Gene (acedb) Vector backbone pGW5 B0511.8 pGN49A pGW9 C01G8.7 pGN49A pGW11 C47B2.3 pGN49A pGW17 B0511.8 pGN59A pGW21 C01G8.7 pGN59A pGW23 C47B2.3 pGN59A

[0121] All the plasmids (pGW-series)are transformed in E. coli AB301-105 (DE3) bacteria by standard methodology. The bacteria are then grown in LB/amp at 37° C. for 14-18 h. These cultures were centrifuged and the bacterial pellet dissolved in S-complete buffer containing 1 mM IPTG and 100 μg/μl ampicillin.

[0122] In 96 well plates containing 100 ξl S-complete (containing 1 mM IPTG and 100 μg/μl ampicillin final concentration) and 10 μl of bacteria solution, 3 nematodes were added at each well, the nematodes were at the L1 growth stage.

[0123] The plates were incubated at 25° C. for 5-6 days. Each day the plates were inspected for development of the larvae and the production of F1 offspring.

[0124] Results

[0125] The RNAi was performed in eight-fold for each constructed plasmid. The results show that when T7 terminators are inserted into the vector backbone, the expected phenotype gives a 100% occurrence. When T7 terminators are not used the reproducibility can decrease up to 50%. As in the previous experiment, the results show that the addition of terminators significantly enhances RNAi performance. DNA fragment B0511.8 B0511.8 C01G8.7 C01G8.7 C47B2.3 C47B2.3 Vector pGN49A pGN59A PGN49A pGN59A pGN49A pGN59A Resulting PGW5 PGW17 PGW9 PGW21 PGW11 PGW23 plasmid Percentage 100 75 100 87.5 100 50 lethal Percentage 0 25 0 12.5 0 50 offspring

[0126] PCR fragment generated by the primers oGN103 and oGN104 on template pCDM8 (SEQ ID NO:20) taccaaggct agcatggttt atcactgata agttgg ataagttggt ggacatatta tgtttatcag tgataaagtg tcaagcatga caaagttgca gccgaataca gtgatccgtg ccggccctgg actgttgaac gaggtcggcg tagacggtct gacgacacgc aaactggcgg aacggttggg ggtgcagcag ccggcgcttt actggcactt caggaacaag cgggcgctgc tcgacgcact ggccgaagcc atgctggcgg agaatcatac gcttcggtgc cgagagccga cgacgactgg cgctcatttc tgatcgggaa tcccgcagct tcaggcaggc ccatgctagc cttggtacca gcacaatgg

[0127] Overlap PCR Fragment, which was used to generate pGN49A (SEQ ID NO:21) gatctggatccggcttactaaaagccagataacagtatgcgtatttgcgc gctgatttttgcggtataagaatatatactgatatgtatacccgaagtat gtcaaaaagaggtgtgctatgaagcagcgtattacagtgacagttgacag cgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtct ggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgct ggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaa atgaacggctcttttgctgacgagaacagggactggtgaaatgcagttta aggtttacacctataaaagagagagccgttatcgtctgtttgtggatgta cagagtgatattattgacacgcccgggcgacggatggtgatccccctggc cagtgcacgtctcttaagcgataaagtctcccgtgaactttacccggtgg tgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagt gtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcga aaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgt caggctcccttatacacagc

[0128] Other aspects of the invention will be clear to the skilled artisan and need not be repeated here. Each reference or publication cited herein is incorporated by reference in its entirety.

[0129] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 21 <210> SEQ ID NO 1 <211> LENGTH: 160 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fragment of pGN1 containing opposable T7 promoters <400> SEQUENCE: 1 ttgtaatacg actcactata gggcgaattc gagctcggta cccggggatc ctctagagtc 60 gaaagcttct cgccctatag tgagtcgtat tacagcttga gtattctata gtgtcaccta 120 aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt 160 <210> SEQ ID NO 2 <211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: DNA sequence containing a T7 terminator <400> SEQUENCE: 2 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttg 49 <210> SEQ ID NO 3 <211> LENGTH: 70 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Oligonucleotide oGN27 <400> SEQUENCE: 3 aattcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct agtgaattct 60 gcagcggtac 70 <210> SEQ ID NO 4 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Oligonucleotide oGN28 <400> SEQUENCE: 4 cgctgcagaa ttcactagca taaccccttg gggcctctaa acgggtcttg aggggttttt 60 tg 62 <210> SEQ ID NO 5 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Oligonucleotide oGN29 <400> SEQUENCE: 5 ctagacgcgt aagcttacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt 60 ttttg 65 <210> SEQ ID NO 6 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Oligonucleotide oGN30 <400> SEQUENCE: 6 agctcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct agtaagctta 60 cgcgt 65 <210> SEQ ID NO 7 <211> LENGTH: 230 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fragment of plasmid pGN9 containing opposable T7 promoters and T7 transcription terminators <400> SEQUENCE: 7 ttgtaatacg actcactata gggcgaattc aaaaaacccc tcaagacccg tttagaggcc 60 ccaaggggtt atgctagtga attctgcagg gtacccgggg atcctctaga cgcgtaagct 120 tactagcata accccttggg gcctctaaac gggtcttgag gggttttttg agcttctcgc 180 cctatagtga gtcgtattac agcttgagta ttctatagtg tcacctaaat 230 <210> SEQ ID NO 8 <211> LENGTH: 3323 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Plasmid pGN9 <400> SEQUENCE: 8 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 60 ggcgaaattg taaacgttaa tattttgtta aaattcgcgt taaatatttg ttaaatcagc 120 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc 180 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac 240 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca 300 cccaaatcaa gttttttgcg gtcgaggtgc cgtaaagctc taaatcggaa ccctaaaggg 360 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag 420 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc 480 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc 540 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag 600 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt 660 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattc aaaaaacccc 720 tcaagacccg tttagaggcc ccaaggggtt atgctagtga attctgcagg gtacccgggg 780 atcctctaga cgcgtaagct tactagcata accccttggg gcctctaaac gggtcttgag 840 gggttttttg agcttctcgc cctatagtga gtcgtattac agcttgagta ttctatagtg 900 tcacctaaat agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 960 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 1020 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 1080 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 1140 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 1200 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 1260 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 1320 gctggcgttt ttcgataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 1380 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 1440 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 1500 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 1560 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 1620 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 1680 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 1740 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 1800 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 1860 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 1920 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 1980 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 2040 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 2100 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 2160 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 2220 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 2280 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 2340 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttggcat 2400 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 2460 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 2520 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 2580 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 2640 gtactcaacc aagtcattct gagaataccg cgcccggcga ccgagttgct cttgcccggc 2700 gtcaatacgg gataatagtg tatgacatag cagaacttta aaagtgctca tcattggaaa 2760 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 2820 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 2880 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 2940 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 3000 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 3060 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa 3120 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct 3180 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag 3240 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc 3300 ggcatcagag cagattgtac tga 3323 <210> SEQ ID NO 9 <211> LENGTH: 3774 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Plasmid pGN29 <400> SEQUENCE: 9 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 60 ggcgaaattg taaacgttaa tattttgtta aaattcgcgt taaatatttg ttaaatcagc 120 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc 180 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac 240 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca 300 cccaaatcaa gttttttgcg gtcgaggtgc cgtaaagctc taaatcggaa ccctaaaggg 360 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag 420 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc 480 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc 540 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag 600 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt 660 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattc aaaaaacccc 720 tcaagacccg tttagaggcc ccaaggggtt atgctagtga attctgcagg gtacccgggg 780 atcctctaga gatccctcga cctcgagatc cattgtgctg gcgcggattc tttatcactg 840 ataagttggt ggacatatta tgtttatcag tgataaagtg tcaagcatga caaagttgca 900 gccgaataca gtgatccgtg ccggccctgg actgttgaac gaggtcggcg tagacggtct 960 gacgacacgc aaactggcgg aacggttggg ggtgcagcag ccggcgcttt actggcactt 1020 caggaacaag cgggcgctgc tcgacgcact ggccgaagcc atgctggcgg agaatcatac 1080 gcttcggtgc cgagagccga cgacgactgg cgctcatttc tgatcgggaa tcccgcagct 1140 tcaggcaggc gctgctcgcc taccgccagc acaatggatc tcgagggatc ttccatacct 1200 accagttctg cgcctgcagg tcgcggccgc gactctctag acgcgtaagc ttactagcat 1260 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gagcttctcg ccctatagtg 1320 agtcgtatta cagcttgagt attctatagt gtcacctaaa tagcttggcg taatcatggt 1380 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg 1440 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt 1500 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 1560 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 1620 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 1680 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 1740 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttcgatagg ctccgccccc 1800 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 1860 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 1920 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 1980 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 2040 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 2100 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 2160 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 2220 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 2280 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 2340 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 2400 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 2460 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 2520 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 2580 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 2640 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 2700 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 2760 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 2820 cagttaatag tttgcgcaac gttgttggca ttgctacagg catcgtggtg tcacgctcgt 2880 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 2940 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 3000 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 3060 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatacc 3120 gcgcccggcg accgagttgc tcttgcccgg cgtcaatacg ggataatagt gtatgacata 3180 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 3240 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 3300 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 3360 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 3420 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 3480 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 3540 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 3600 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 3660 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 3720 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctga 3774 <210> SEQ ID NO 10 <211> LENGTH: 5148 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Plasmid pGN39 <400> SEQUENCE: 10 taatacgact cactataggg cgaattcaaa aaacccctca agacccgttt agaggcccca 60 aggggttatg ctagtgaatt ctgcagcggt acccggggat cctctagaga tccctcgacc 120 tcgagatcca ttgtgctgga aagatcacaa gtttgtacaa aaaagctgaa cgagaaacgt 180 aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag actacataat 240 actgtaaaac acaacatatc cagtcactat ggcggccgca ttaggcaccc caggctttac 300 actttatgct tccggctcgt ataatgtgtg gattttgagt taggatccgg cgagattttc 360 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc 420 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa 480 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa 540 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattccg 600 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt 660 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg 720 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt 780 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac 840 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg 900 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc 960 cgtctgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga 1020 gtggcagggc ggggcgtaaa gatctggatc cggcttacta aaagccagat aacagtatgc 1080 gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat 1140 gtcaaaaaga ggtgtgctat gaagcagcgt attacagtga cagttgacag cgacagctat 1200 cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga 1260 atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg 1320 aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg gactggtgaa 1380 atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta 1440 cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt 1500 ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc 1560 tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg 1620 gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga 1680 atataaatgt caggctccct tatacacagc cagtctgcag gtcgaccata gtgactggat 1740 atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt 1800 gatatttata tcattttacg tttctcgttc agctttcttg tacaaagtgg tgatctttcc 1860 agcacaatgg atctcgaggg atcttccata cctaccagtt ctgcgcctgc aggtcgcggc 1920 cgcgactcta gacgcgtaag cttactagca taaccccttg gggcctctaa acgggtcttg 1980 aggggttttt tgagcttctc gccctatagt gagtcgtatt acagcttgag tattctatag 2040 tgtcacctaa atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 2100 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 2160 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 2220 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 2280 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 2340 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 2400 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 2460 ttgctggcgt ttttcgatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 2520 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 2580 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 2640 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 2700 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 2760 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 2820 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 2880 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 2940 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 3000 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 3060 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 3120 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 3180 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 3240 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 3300 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 3360 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 3420 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 3480 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttggc 3540 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 3600 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 3660 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 3720 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 3780 gagtactcaa ccaagtcatt ctgagaatac cgcgcccggc gaccgagttg ctcttgcccg 3840 gcgtcaatac gggataatag tgtatgacat agcagaactt taaaagtgct catcattgga 3900 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 3960 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 4020 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 4080 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 4140 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 4200 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 4260 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac 4320 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc 4380 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat 4440 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga 4500 tgcgtaagga gaaaataccg catcaggcga aattgtaaac gttaatattt tgttaaaatt 4560 cgcgttaaat atttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat 4620 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa 4680 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg 4740 cgatggccca ctacgtgaac catcacccaa atcaagtttt ttgcggtcga ggtgccgtaa 4800 agctctaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc 4860 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag 4920 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg 4980 cgcgtccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 5040 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 5100 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattg 5148 <210> SEQ ID N O 11 <211> LENGTH: 3715 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Plasmid TopoRNAi <400> SEQUENCE: 11 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 60 ggcgaaattg taaacgttaa tattttgtta aaattcgcgt taaatatttg ttaaatcagc 120 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc 180 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac 240 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca 300 cccaaatcaa gttttttgcg gtcgaggtgc cgtaaagctc taaatcggaa ccctaaaggg 360 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag 420 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc 480 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc 540 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag 600 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt 660 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattc aaaaaacccc 720 tcaagacccg tttagaggcc ccaaggggtt atgctagtga attctgcagg gtacccgggg 780 atcctctaga gatccctcga cctcgagatc cattgtggtg gaattctacc aaggctagca 840 tgggcagccg aatacagtga tccgtgccgg ccctggactg ttgaacgagg tcggcgtaga 900 cggtctgacg acacgcaaac tggcggaacg gttgggggtg cagcagccgg cgctttactg 960 gcacttcagg aacaagcggg cgctgctcga cgcactggcc gaagccatgc tggcggagaa 1020 tcatacgctt cggtgccgag agccgacgac gactggcgct catttctgat cgggaatccc 1080 gcagccatgc tagccttggt agaattccac cacaatggat ctcgagggat cttccatacc 1140 taccagttct gcgcctgcag gtcgcggccg cgactctcta gacgcgtaag cttactagca 1200 taaccccttg gggcctctaa acgggtcttg aggggttttt tgagcttctc gccctatagt 1260 gagtcgtatt acagcttgag tattctatag tgtcacctaa atagcttggc gtaatcatgg 1320 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 1380 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 1440 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 1500 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 1560 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 1620 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 1680 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttcgatag gctccgcccc 1740 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 1800 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 1860 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 1920 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 1980 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 2040 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 2100 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 2160 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 2220 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 2280 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 2340 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 2400 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 2460 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 2520 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 2580 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 2640 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 2700 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 2760 ccagttaata gtttgcgcaa cgttgttggc attgctacag gcatcgtggt gtcacgctcg 2820 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 2880 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 2940 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 3000 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatac 3060 cgcgcccggc gaccgagttg ctcttgcccg gcgtcaatac gggataatag tgtatgacat 3120 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 3180 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 3240 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 3300 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 3360 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 3420 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 3480 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 3540 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc 3600 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt 3660 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actga 3715 <210> SEQ ID NO 12 <211> LENGTH: 4107 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Plasmid pGN49A <400> SEQUENCE: 12 tgtaatacga ctcactatag ggcgaattca aaaaacccct caagacccgt ttagaggccc 60 caaggggtta tgctagtgaa ttctgcagcg gtacccgggg atcctctaga gatccctcga 120 cctcgagatc cattgtgctg gaaaggatct ggatccggct tactaaaagc cagataacag 180 tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga 240 agtatgtcaa aaagaggtgt gctatgaagc agcgtattac agtgacagtt gacagcgaca 300 gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat 360 gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc aggaagggat 420 ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga acagggactg 480 gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg 540 atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg 600 cacgtctctt aagcgataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg 660 aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag 720 aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct 780 ggggaatata aatgtcaggc tcccttatac acagcctttc cagcacaatg gatctcgagg 840 gatcttccat acctaccagt tctgcgcctg caggtcgcgg ccgcgactct agacgcgtaa 900 gcttactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgagcttct 960 cgccctatag tgagtcgtat tacagcttga gtattctata gtgtcaccta aatagcttgg 1020 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 1080 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 1140 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 1200 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 1260 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 1320 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 1380 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttcgata 1440 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 1500 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 1560 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 1620 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 1680 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 1740 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 1800 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 1860 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 1920 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 1980 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 2040 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 2100 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 2160 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 2220 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 2280 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 2340 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 2400 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 2460 taagtagttc gccagttaat agtttgcgca acgttgttgg cattgctaca ggcatcgtgg 2520 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 2580 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 2640 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 2700 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 2760 tctgagaata ccgcgcccgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 2820 gtgtatgaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 2880 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 2940 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 3000 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 3060 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 3120 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 3180 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga 3240 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc 3300 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg 3360 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg 3420 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 3480 gcatcaggcg aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tatttgttaa 3540 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 3600 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 3660 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 3720 ccatcaccca aatcaagttt tttgcggtcg aggtgccgta aagctctaaa tcggaaccct 3780 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 3840 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 3900 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca 3960 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg 4020 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 4080 gacgttgtaa aacgacggcc agtgaat 4107 <210> SEQ ID NO 13 <211> LENGTH: 4001 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Plasmid pGN59A <400> SEQUENCE: 13 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 60 ggcgaaattg taaacgttaa tattttgtta aaattcgcgt taaatatttg ttaaatcagc 120 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc 180 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac 240 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca 300 cccaaatcaa gttttttgcg gtcgaggtgc cgtaaagctc taaatcggaa ccctaaaggg 360 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag 420 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc 480 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc 540 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag 600 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt 660 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattc gagctcggta 720 cccggggatc ctctagagat ccctcgacct cgagatccat tgtgctggaa aggatctgga 780 tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt ttgcggtata 840 agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtgtgct atgaagcagc 900 gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat atgatgtcaa 960 tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg 1020 ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg aaatgaacgg 1080 ctcttttgct gacgagaaca gggactggtg aaatgcagtt taaggtttac acctataaaa 1140 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgcccgggc 1200 gacggatggt gatccccctg gccagtgcac gtctcttaag cgataaagtc tcccgtgaac 1260 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca 1320 gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca 1380 tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc cttatacaca 1440 gcctttccag cacaatggat ctcgagggat cttccatacc taccagttct gcgcctgcag 1500 gtcgcggccg cgactctcta gagtcgaaag cttctcgccc tatagtgagt cgtattacag 1560 cttgagtatt ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc 1620 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 1680 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 1740 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 1800 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 1860 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 1920 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 1980 ccgtaaaaag gccgcgttgc tggcgttttt cgataggctc cgcccccctg acgagcatca 2040 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 2100 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 2160 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 2220 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 2280 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 2340 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 2400 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 2460 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 2520 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 2580 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 2640 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 2700 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 2760 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 2820 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 2880 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 2940 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 3000 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 3060 gcgcaacgtt gttggcattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 3120 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 3180 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 3240 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 3300 cttttctgtg actggtgagt actcaaccaa gtcattctga gaataccgcg cccggcgacc 3360 gagttgctct tgcccggcgt caatacggga taatagtgta tgacatagca gaactttaaa 3420 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 3480 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 3540 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 3600 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 3660 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 3720 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat 3780 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg 3840 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta 3900 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg 3960 gggctggctt aactatgcgg catcagagca gattgtactg a 4001 <210> SEQ ID NO 14 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Oligonucleotide oGN103 <400> SEQUENCE: 14 taccaaggct agcatggttt atcactgata agttgg 36 <210> SEQ ID NO 15 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Olgigonucleotide oGN104 <400> SEQUENCE: 15 taccaaggct agcatgggcc tgcctgaagg ctgc 34 <210> SEQ ID NO 16 <211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Oligonucleotide oGN126 <400> SEQUENCE: 16 gatctggatc cggcttacta aaagccagat aacagtatgc 40 <210> SEQ ID NO 17 <211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Oligonucleotide oGN127 <400> SEQUENCE: 17 ggagacttta tcgcttaaga gacgtgcact ggccaggggg atcacc 46 <210> SEQ ID NO 18 <211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Oligonucleotide oGN128 <400> SEQUENCE: 18 ccagtgcacg tctcttaagc gataaagtct cccgtgaact ttacccggtg g 51 <210> SEQ ID NO 19 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Oligonucleotide oGN129 <400> SEQUENCE: 19 gctgtgtata agggagcctg acatttatat tccccag 37 <210> SEQ ID NO 20 <211> LENGTH: 375 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: PCR fragment generated by primers oGN103 and 0GN104 on pCDM8 <400> SEQUENCE: 20 taccaaggct agcatggttt atcactgata agttggataa gttggtggac atattatgtt 60 tatcagtgat aaagtgtcaa gcatgacaaa gttgcagccg aatacagtga tccgtgccgg 120 ccctggactg ttgaacgagg tcggcgtaga cggtctgacg acacgcaaac tggcggaacg 180 gttgggggtg cagcagccgg cgctttactg gcacttcagg aacaagcggg cgctgctcga 240 cgcactggcc gaagccatgc tggcggagaa tcatacgctt cggtgccgag agccgacgac 300 gactggcgct catttctgat cgggaatccc gcagcttcag gcaggcccat gctagccttg 360 gtaccagcac aatgg 375 <210> SEQ ID NO 21 <211> LENGTH: 670 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: PCR fragment <400> SEQUENCE: 21 gatctggatc cggcttacta aaagccagat aacagtatgc gtatttgcgc gctgattttt 60 gcggtataag aatatatact gatatgtata cccgaagtat gtcaaaaaga ggtgtgctat 120 gaagcagcgt attacagtga cagttgacag cgacagctat cagttgctca aggcatatat 180 gatgtcaata tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt 240 gccgaacgct ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa 300 atgaacggct cttttgctga cgagaacagg gactggtgaa atgcagttta aggtttacac 360 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac 420 gcccgggcga cggatggtga tccccctggc cagtgcacgt ctcttaagcg ataaagtctc 480 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga 540 tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga 600 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt caggctccct 660 tatacacagc 670 

We claim:
 1. A DNA construct comprising: a) a first promoter and b) a second promoter, in which the first and second promoter are in opposite orientation to each other and define: c) an inter-promoter region positioned downstream of the 3′ end of the first promoter and downstream of the 3′ end of the second promoter; and which DNA construct further comprises: d) at least one cloning site positioned in the inter-promoter region; and e) a first transcription terminator, positioned (as seen from the 3′ end of the first promoter) downstream of the first promoter and downstream of the at least one cloning site, wherein the first transcription terminator is operably linked to the first promoter.
 2. A DNA construct according to claim 1, further comprising: f) a second transcription terminator positioned (as seen from the 3′ end of the second promoter) downstream of the second promoter and downstream of the at least one cloning site. wherein the second transcription terminator is operably linked to the second promoter.
 3. A DNA construct according to claim 1 or 2, in which the first transcription terminator is positioned in the inter-promoter region.
 4. A DNA construct according to claim 1 or 2, in which the first transcription terminator is positioned (as seen from the 3′ end of the first promoter) downstream of the first promoter, downstream of the at least one cloning site, and downstream of the 5′ end of the second promoter.
 5. A DNA construct according to any one of claims 2, 3 or 4, in which the second transcription terminator is positioned in the inter-promoter region.
 6. A DNA construct according to any of claims 2, 3 or 4 in which the second transcription terminator is positioned (as seen from the 3′ end of the second promoter) downstream of the second promoter, downstream of the at least one cloning site, and downstream of the 5′ end of the first promoter.
 7. A DNA construct according to any one of claims 4, 5 or 6, in which the 3′ end of the first transcription terminator is separated from the 5′ end of the second promoter by no more than 2000 nucleotides, preferably no more than 1000 nucleotides, more preferably no more than 500 nucleotides, even more preferably no more than 200 nucleotides, especially preferably no more than 100 nucleotides, more especially preferably no more than 50 nucleotides, even more especially preferably no more than 20 nucleotides, particularly preferably no more than 10 nucleotides, more particularly preferably no more than 6 nucleotides.
 8. A DNA construct according to any one of claims 6 or 7, in which the 3′ end of the second transcription terminator is separated from the 5′ end of the first promoter by no more than 2000 nucleotides, preferably no more than 1000 nucleotides, more preferably no more than 500 nucleotides, even more preferably no more than 200 nucleotides, especially preferably no more than 100 nucleotides, more especially preferably no more than 50 nucleotides, even more especially preferably no more than 20 nucleotides, particularly preferably no more than 10 nucleotides, more particularly preferably no more than 6 nucleotides.
 9. A construct according to any one of the preceding claims wherein the first and the second promoter are identical.
 10. A DNA construct according to any one of the claims 1 to 7 wherein the first and the second promoter are non-identical.
 11. A DNA construct according to claims 8 or 9 wherein the first promoter and the second promoter are independently chosen from T7, T3 or SP6 promoters.
 12. A construct according to any one of the preceding claims wherein the cloning site comprises at least one restriction site.
 13. A DNA according to claim 11 wherein the cloning site comprises at least two restriction sites flanking a sequence of stuffer DNA.
 14. A DNA construct according to claim 12 wherein the at least two restriction sites are identical.
 15. A DNA construct according to any one of the claims 12 to 13 wherein the at least one restriction site or the at least two restriction sites restriction sites are BstXI sites.
 16. A DNA construct according to any one of the claims 12 to 13 wherein the restriction sites are XcmI sites.
 17. A DNA construct according to any one of the preceding claims wherein the cloning site further comprises attR1 and attR2 recombination sequences.
 18. A DNA construct according to any one of the preceding claims wherein the cloning site further comprises a ccdB nucleotide sequence.
 19. A DNA construct according to claim 17 wherein the ccdB nucleotide sequence further comprises at least one unique restriction site.
 20. A DNA construct according to claim 18 wherein the at least one unique restriction site are blunt-end restriction sites.
 21. A DNA construct according to claim 19 wherein the blunt-end restriction sites are SrfI sites.
 22. A DNA according to any one of the preceding claims which further comprises: g) a DNA fragment inserted in the at least one cloning site.
 23. A DNA construct according to any one of the preceding claims which is a plasmid or vector.
 24. A plasmid or vector as claimed in claim 23 having the nucleotide sequence illustrated in FIG. 10, FIG. 11, FIG. 12, FIG. 13, FIG. 14, or FIG.
 15. 25. Use of the DNA construct according to any one of the preceding claims for the production of double-stranded RNA for RNA inhibition.
 26. A bacterial strain harbouring the DNA construct according to any one of the preceding claims.
 27. A bacterial strain according to claim 26, wherein said bacteria strain is an E. coli strain.
 28. Use of the bacterial strain according to claims 26 or 27 for the production of double-stranded RNA for RNA inhibition. 